TthPlus™ DNA polymerase is isolated from the Thermus thermophilus strain. TthPlus™ DNA polymerase is a single 92 kDa polypeptide showing a 5'-3' exonuclease activity but lacking 3'-5' exonuclease activity. It catalyzes the polymerization of nucleotides into double-stranded DNA in the presence of MgCl2 . Its efficiancy has been shown more particularly on large DNA fragments up to 12 kb (using lambda phage DNA as a template). TthPlus™ DNA polymerase is also capable of catalyzing the polymerization of DNA using a RNA template in the presence of MnCl2 . The ability of TthPlus™ DNA polymerase to reverse transcribe at elevated temperatures (70°C) minimizes the problems encountered with strong secondary structures in RNA since they are unstable at higher reaction temperatures. Higher temperatures also result in increased specificy of primer hybridization and extension. In coupled RT/PCR assays, TthPlus™ is about 50-100 times more efficient than Taq DNA polymerase.
TthPlus™ DNA polymerase is delivered with 10x RT-buffer, 5x amplification-buffer and separate MnCl2 (25 mM) and MgCl2 (50 mM) solutions. The 5x amplification-buffer contains EGTA, which binds/neutalizes Mn2+ from the RT-reaction. Therefore after RT it is not necessary to change the buffers. For subsequent ampli-fication we recommend to use BioTherm™ or KlenTherm™ DNA polymerase.
- DNA polymerase activity in the presence of MgCl2
- reverse transcriptase activity in the presence of MnCl2
- reverse transcription at elevated temperature minimising secondary structure problems
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 minutes at 72°C under the following reaction conditions: 25 mM TAPS buffer (Tris-(hydroxymethyl)-methyl- amino-propanesulfonic acid, sodium salt) pH 9.3 (25°C), 50 mM KCl, 2 mM MgCl2 , 1 mM .-mer-captoethanol, 200 µM dNTPs and 10 µg of calf thymus DNA in a final reaction volume of 50 µl.
10 mM K-phosphate buffer pH 7.0 (25°C), 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 50% glycerol (v/v), 0,1 mg/ml BSA
Store TthPlus™ DNA polymerase, preferably at -20°C, in a constant temperature freezer.
10X REACTION BUFER
670 mM Tris-HCl pH 8.8 (25°C), 166 mM (NH4)2SO4, 0.1% Tween 20
5x AMPLIFICATION BUFFER
335 mM Tris-HCl pH 8.8 (25°C), 83 mM (NH4)2SO4, 3.75 mM EGTA, 25% glycerol (v/v), 0.1% Tween 20
25 mM MnCl2
50 mM MgCl2
The optimal experimental conditions depend on the system used and they should be individually determined. The Mg2+ or Mn2+ concentrations and the enzyme amount are the limiting factors for an accurate result. Tra-ditionally 5 units of enzyme and a MnCl2 concentration of 1 mM are used for the reverse transcription in a final 50 µl reaction volume. For the amplification 2.5 MgCl2 concentration of 1.5 mM are used for a final reaction volume of 50 µl.
GeneScript™ reverse transcriptase, BioTherm™ DNA polymerase, KlenTherm™ DNA Polymerase, dNTPs
Comparison of sensitivity of RT-PCR with TthPlus™ DNA polymerase and MMLV-RT
Comparison of RT-PCR with TthPlus™ DNA polymerase and MMLV-RT in 16S-rRNA system
10X ONE-TUBE BUFFER
500 Mm bicine-KOH pH 8.3, 1 M KOAc pH 7.5, 30% glycerol
50 mM Mn/OAc)2
VARIOUS CONDITIONS FOR RT-PCR
Two different buffer system can be used for RT-PCR with TthPlus DNA polymerase. The first system for Tth DNA polymerase consists of 4 buffers: 1. reverse transciption (RT) buffer, 2. PCR (amplification buffer), 3. MnCl2 (supplement for RT buffer), 4. MgCl2 (supplement for PCR buffer). The reaction has to be carried out in two steps: RT and PCR in two different vials.
The second buffer system is so-called one-tube buffer (10x) for one-step RT-PCR. Both reactions (RT and PCR) are carried out in the same buffer and the same vial. The one-tube buffer does not contain Mn(OAc)2. Mn(OAc)2 is provided extra and have to be added to the one-tube buffer before the experiment. The protocol to use our TthPlus DNA polymerase is described below (buffer and polymerase conditions and cycle conditions). It was worked out for real-time RT-PCR by our customer Roboscreen GmbH.
- Ruttiman C. Cotara S.M., Zaldivar J. and Vicuna R. (1985) DNA polymerase from the extremly thermophylic bacterium Thermus thermophilus HB-8. Eur. J. Biochemistry, 149, 41-46
- Myers T.W. and Gelfand D.H. (1991)Reverse transcription and DNA amplification by a Thermus thermo-philus DNA polymerase. Biochemistry, 30, 7661-7666